NaK-ATPase is an ubiquitous, membrane-bound ATP-dependent enzyme complex which mediates the active transport of Na+ and K+ across the plasma membrane in all cells in which maintenance of cation concentration gradients is a necessary prerequisite for cell homostasis. The modulation of this transport enzyme is involved in a broad spectrum of clinical problems including the mechanisms of cardiac glycoside action, hypertension, obesity, cystic fibrosis, and red cell membrane diseases. The understanding of the detailed molecular structure of this biologically important enzyme system is necessary in order to elucidate structural/functional correlations at the protein level and to relate these findings to normal and pathological processes, particularly those involving the cardiovascular system. The general goal of this proposal is to elucidate the structure and regulation of the NaK-ATPase using the newly developed techniques of recombinant DNA. The duck salt gland is themodel of choice due to the high levels of NaK-ATPase present (up to 40% of microsomal protein content), ease and magnitude of induction and its known biochemical characteristics including molecular weight of each subunit, amino acid composition, NH2-terminal amino acid sequence and availability of antibody to each subunit. The specific objectives are: (1) To construct, identify and characterize cDNA clones containing the sequences for each subunit of duck salt gland NaK-ATPase. The process of identifying the recombinant NaK-ATPase clones will include differential RNA blot hybridization, in vitro cell-free translation, immunoprecipitation, and synthetic oligonucleotide probes constructed from the known NH2-terminal amino acid sequence. (2) To analyze the nucleotide and deduced amino sequence of the NaK-ATPase cDNA clones using standard techniques. (3) To determine the number of NaK-ATPase genes and (4) tissue/developmental specificity of the enzyme by restriction mapping of the clones followed by cDNA probing of the DNA blots. (5) To analyze the evolutionary conservation of the NaK-ATPase sequences by isolating DNA from sources representing different phyla. The probe isolated in the course of these experiments may prove useful in isolating the corresponding human sequences. Study of the human NaK-ATPase cDNA is the long term objective.